Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

Article abstract of DOI:10.1016/j.bmcl.2009.02.017

Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of in

Full text of DOI:10.1016/j.bmcl.2009.02.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4882–4884
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase
selectivity and cell potency
*
*
David R. Anderson , Marvin J. Meyers , Ravi G. Kurumbail, Nicole Caspers, Gennadiy I. Poda, Scott A. Long,
Betsy S. Pierce, Matthew W. Mahoney, Robert J. Mourey, Mihir D. Parikh
Pfizer Global Research and Development, St. Louis Laboratories, 700 Chesterfield Parkway W, Chesterfield, MO 63017, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with
potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized
by examination of X-ray crystal structures of inhibitors bound to MK2.
Received 6 October 2008
Revised 3 February 2009
Accepted 5 February 2009
Available online 8 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
MK2
MAPKAP-K2
Kinase inhibitor
In Part 1 of this Letter we described a new class of benzothio-
phene inhibitors of mitogen activated protein kinase-activated
protein kinase 2 (MK2). While these compounds were potent
inhibitors of MK2 with sub-micromolar cellular potencies, selectiv-
ity against other kinases, including CDK2, was not achieved. In this
Letter, strategies used to improve the selectivity of this class of
inhibitors are described.
Selectivity
Element
Hinge-binding
Element
R
HN
NH
O
HN
O
NH
S
O
N
2
Previously we had disclosed that placing a rigid group near the
hinge binding element resulted in remarkable selectivity enhance-
ment for a different chemical class of MK2 inhibitors.1 For example,
compound 1 was found to possess good selectivity against a num-
ber of kinases, and this selectivity was attributed to the rigid aryl
group attached to the 2-position of the pyridine. Based on this
precedent, we sought to modify the hinge binding element in the
benzothiophene class (e.g., 2) to provide an attachment point for
this selectivity element as shown in 3.
1
Hinge-binding
Element
R
HN
NH
X
To assess this hypothesis, compounds were prepared with an
additional ring fused to the benzothiophene ring, which would
provide a rigid attachment point for a selectivity element. To verify
that the hetero atom, as part of an additional fused ring, could be
an effective hinge-binding element, compounds without the selec-
tivity element were first prepared (Fig. 1).
The synthesis of furan and dihydro–furan analogs is shown in
Scheme 1. Chlorobenzothiophene 42 was demethylated with BBr3
and alkylated with allyl bromide to provide 5. Heating 5 to
200 °C in diethylaniline regioselectively provided 6 in quantitative
S
O
3
Figure 1. Rationale for improving selectivity of benzothiophene inhibitors of MK2.
yield. Treatment of 6 with OsO4/NaIO4 gave a tautomeric mixture
of 7 and 8. This mixture was converted to furan 9 by dehydration
in phosphoric acid. Dihydrofuran 10 was prepared by sodium boro-
hydride reduction, mesylate formation of the resulting primary
alcohol and cyclization with sodium bicarbonate. Chlorobenzothi-
ophenes 9 and 10 were then elaborated as described in Part 1 of
this series by Buchwald coupling with 11, followed by deprotection
and cyclization to afford diazapenes 12 and 13.
* Corresponding authors. Tel.: +1 636 247 7651 (D.R.A.).
E-mail address: david.r.anderson@pfizer.com (D.R. Anderson).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.017

D. R. Anderson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4882–4884
4883
HO
O
Cl
Cl
Cl
O
Cl
c
O
a,b
O
d
HO
O
O
O
S
O
S
O
S
O
S
4
O
5
6
7
Cl
HN
S
O
O
g
e
f
O
NH
O
HO
Cl
O
S
O
O
9
S
O
12
8
HN
Cl
g
O
O
O
NH
NHBoc
H2N
S
S
O
O
10
13
11
Scheme 1. Reagents and conditions: (a) BBr3; (b) allyl bromide, K2CO3, DMF; (c) PhNEt2, 200 °C; (d) OsO4/NaIO4; (e) H3PO4, 120 °C; (f) (i) NaBH4; (ii) MsCl; (iii) NaHCO3; (g) (i)
(R)-propane-1,2-diamine, Pd2(dba)3 (5 mol %), ( )BINAP (10 mol %), Cs2CO3 (2 equiv) toluene, 110 °C, 24 h; (ii) TFA/CH2Cl2; (iii) NaOMe/MeOH.
The synthesis of an analog with a pyridine-containing binding
element is shown in Scheme 2. Nitroquinoline 14 was treated with
ethyl cyanoacetate and KOH in DMF to provide aminocyanoquino-
line 15.3 The amino group was converted to bromide via diazotiza-
tion to provide 16. Nucleophilic aromatic substitution on the
resulting bromide with methyl thioglycolate and subsequent thio-
phene formation produced 17. Conversion of the amino group in 17
to bromide 18 was accomplished by diazotization and substitution.
The resulting bromide was then elaborated as described for 9 and
10 to produce diazapene 19.
Table 1
MK2 and CDK2 potencies of analogs 2, 12–13, 19
Compound
number
MK2 inhibition
CDK2 inhibition
U937 TNFa release
IC50 (lM)
a
a
a
IC50
(l
M)
IC50
(lM)
2
0.04
0.012
0.001
0.005
0.7
12
13
19
0.016
0.028
0.001
0.09
0.26
0.05
0.0008
a
Values are means of at least three experiments and standard deviations were
within 50% of the reported value Assays conditions are the same as described in Ref. 1.
The potencies of the analogs prepared in Schemes 1 and 2 for
MK2, CDK2 and TNF
a production in LPS-stimulated U937 cells
are reported in Table 1. All three hinge binding replacements are
potent MK2 and CDK2 inhibitors. Furan analog 12 and pyridine
analog 19 in particular were found to be exceptionally potent
inhibitors of MK2 and had IC50 values of less than 100 nM in the
cell assay. Broad panel kinase selectivity screening of 12 was con-
were prepared. The synthesis of these analogs is shown in Scheme
3. Propargyl alcohol (20) was converted to allyl bromide 21 and
was used in the alkylation as described previously in the conver-
sion of 4 to 5. The remaining sequence of rearrangement, oxidative
cleavage of the alkene and dehydration produced 24. Analog 19
was doubly protected with boc groups and the pyridine oxidized
to form 25. Reaction with oxalyl chloride and removal of the boc
protecting groups provided intermediate 26. Suzuki coupling with
phenyl boronic acid yielded compound 27.
ducted. Potencies for a total of 109 kinases were evaluated at 1
concentration and 26 kinases (24%) showed >70% inhibition.
lM
In an attempt to improve the selectivity of these inhibitors for
MK2, compounds with aromatic substituents in the 2-position
The potencies of these analogs for MK2, CDK2 and in cell based
TNFa assay are shown in Table 2. Substitution of the furan ring did
CN
not improve selectivity for MK2 over CDK2 and a loss of overall po-
tency was observed for both kinases. Similarly, a loss of cellular po-
tency was also observed. Broad panel kinase selectivity screening
of 24 was conducted. A total of 126 kinases were profiled at
NO2
NH2
a
N
N
15
14
1
l
M concentration and 14 kinases (11%) showed >70% inhibition.
b
Substitution at the pyridine ring was less detrimental to po-
CN
tency. Less than a 10-fold drop in MK2 potency was observed com-
paring 19 to 27; however the ratio of MK2 to CDK2 potency was
improved approximately 20-fold. This improvement in selectivity
may be rationalized by the different trajectories of the aromatic
ring emanating from a 5-versus a 6-membered ring. Furthermore,
NH2
c
Br
N
CO2Me
S
N
17
16
HN
the cell potency was well below 1 lM. Compound 26 had MK2 po-
d
tency and CDK2 selectivity intermediate between 19 and 27 sug-
gesting that the size of the 2-substituent may play a role in both
potency and selectivity.
Intermediate 26 provided a convenient late stage intermediate
for facile analog synthesis via Suzuki coupling. Results are summa-
rized in Table 3.
The analogs described in Table 3 are within approximately 10-
fold in potency for MK2 indicating that most substitutions are well
tolerated. Many analogs have improved selectivity ratios compared
Br
e
N
N
NH
CO2Me
S
O
S
18
19
Scheme 2. Reagents and conditions: (a) (i) ethyl cyanoacetate, KOH, DMF; (ii) HCl;
(b) (i) NaNO2; (ii) HBr; (c) Methyl mercaptoacetate, NaOMe, MeOH; (d) t-BuONO,
CuBr2; (e) (i) (R)-propane-1,2-diamine, Pd2(dba)3 (5 mol %), ( )BINAP (10 mol %),
Cs2CO3 (2 equiv) toluene, 110 °C, 24 h; (ii) TFA/CH2Cl2; (iii) NaOMe/MeOH.

4884
D. R. Anderson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4882–4884
a
Br
OH
b, c, d
g, h
20
21
Ph
Ph
Cl
Ph
Cl
HN
S
HO
e
O
f
O
O
O
4
NH
S
S
O
O
O
22
23
24
Boc
Cl
Ph
N
HN
HN
i, j
N+
k
19
-O
Boc
NH
N
N
N
NH
S
O
S
O
S
O
25
26
27
Scheme 3. Reagents and conditions: (a) (i) PhMgBr, CuI; (ii) PPh3, CBr4; (b) BBr; (c) K2CO3; (d) PhNEt2, 200 ° C; (e) (i) OsO4, NaOI4; (ii) H3PO4, 120 °C; (f) (i) (R)-propane-1,2-
diamine, Pd2(dba)3 (5 mol %), ( )BINAP (10 mol %), Cs2CO3 (2 equiv), toluene, 110 °C, 24 h; (ii) TFA/CH2Cl2; (iii) NaOMe/MeOH; (g) Boc2O, DMAP, TEA; (h) m-CPBA (i) (COCl)2,
DMF; (j) HCl; (k) PhB(OH)2, Pd(PPh3)4, Na2CO3, 80 °C.
Table 2
MK2 and CDK2 potencies of analogs 24, 26–27
Compound
number
MK2 inhibition
CDK2 inhibition
U937 TNFa release
IC50 (lM)
a
a
a
IC50
(
lM)
IC50
(lM)
24
26
27
0.15
0.002
0.009
0.25
0.025
0.2
6.8
0.13
0.18
a
Values are means of at least three experiments and standard deviations were
within 50% of the reported value. Assays conditions are the same as described in
Ref. 1.
to 27, however, indicating that substitution in this region has an
impact on selectivity. Compounds 29 and 35 have cell potency val-
ues below 500 nM and have >1000-fold selectivity for MK2 versus
CDK2.
Figure 2. X-ray crystal structure of 35 in MK2 (3.7 Å resolution 3FYJ).
To better understand the potency and selectivity of these com-
pounds, a crystal structure of 35 in MK2 was obtained at 3.7 Å and
is shown in Figure 2. While this is a low resolution crystal struc-
ture, some analysis may be made. The lactam carbonyl interacts
with the conserved Lys93 and the Asp207 of the activation loop.
The 3-pyridyl nitrogen appears to make no meaningful interaction
with MK2, but the group occupies the same space as the previously
described selective series of MK2 inhibitors.
The selectivity of 29 and 35 was investigated by screening
against a subset panel of kinases comprised of targets that previous
series of compounds had inhibitory activity against. Of the 50 ki-
nases targeted for screening, only 4 had >70% inhibition at 1
(AMPK, PIM1, MEKK5 and BRSK1).
lM
The mechanism of action within the cell based assay was as-
sessed by comparing inhibition of phosphorylation of Ser78 on
HSP27 within the cell to TNF
ously.1 The IC50 values for phosphorylated HSP27 were within 2
fold of TNF IC50 values for both of these inhibitors with no effect
a suppression as described previ-
Table 3
a
MK2 and CDK2 potencies of analogs 28–35
on levels of phospho-p38 or phospho-JNK2. The correlation be-
tween a target biomarker and suppression of cytokine release indi-
Compound
number
R
MK2 inhibition
CDK2
U937 TNFa
a
IC50
(lM)
inhibition
release
cates that both 29 and 35 are inhibiting TNFa expression by
a
a
IC50
(
l
M)
IC50
(l
M)
selectively inhibiting MK2.
28
29
30
31
4-Pyridyl
3-Pyridyl
5-Pyrimidinyl
2-
Methoxyphenyl
3-
Methoxyphenyl
2-Fluorophenyl 0.014
2-
0.014
0.005
0.023
0.020
0.39
6.33
10.8
2.73
0.74
0.22
0.64
1.04
In summary, we have discovered a new, potent and selective
class of MK2 inhibitors that have less than 500 nM potency in a
cell-based assay. Selectivity was improved by modification of the
hinge binding element into a ring to provide a scaffold for the re-
quired selectivity element. The in vivo pharmacology of these
inhibitors will be described in a future communication.
32
0.041
2.04
0.69
33
34
10.9
2.21
0.93
3.36
0.03
References and notes
Methylphenyl
4-Methyl-3-
pyridyl
35
0.005
7.92
0.15
1. Anderson, D. R.; Meyers, M. J.; Vernier, W. F.; Mahoney, M. W.; Kurumbail, R. G.;
Caspers, N.; Poda, G. I.; Schindler, J. F.; Reitz, D. B.; Mourey, R. J. J. Med. Chem.
2007, 50, 2647.
2. Higa, T.; Krubsack, A. J. J. Org. Chem. 1976, 41, 3399.
3. Tomioka, Y.; Ohkubo, K.; Motoyoshi, Y. Chem. Pharm. Bull. 1985, 33, 1360.
a
Values are means of at least three experiments and standard deviations were
within 50% of the reported values. Assays conditions are the same as described in
Ref. 1.